The overall objective is to extend our knowledge of macromolecular structure-function relationships by reducing the time and effort required to determine three-dimensional structures of biological molecules. The research focuses on protein crystallography and is aimed at developing two new phasing techniques to the point where their potentials can be fully exploited. One of the techniques, direct methods with anomalous scattering, looks promising and was recently used in the structure determination of Cd,Zn metallothionein. The other technique, direct methods with single isomorphous replacement, looks like it will be even more powerful. In both techniques, the general theory (developed by Nobel Laureate Herbert Hauptman) has been confirmed and implemented in a rudimentary fashion, but several hurdles remain before the procedures can be used routinely. The specific aims of this proposal are designed to overcome those hurdles and are as follows: (1) to establish the limits of applicability of the new procedures; (2) to complete the direct methods phasing process by implementing and evaluating procedures for determining individual phases from the values predicted by the new theories; (3) to analyze and evaluate results from the only practical application of the direct methods-anomalous scattering procedure to date; (4) to evaluate the new direct methods-single isomorphous refinement theory and make the first application with real (experimental) data; (5) to examine ways of integrating the new procedures with existing phasing methods; (6) to develop an efficient, exportable computer program system for carrying out the procedures in an automated fashion, and to provide feedback regarding practical implementation problems. The new techniques have several advantages over existing methods. They should provide powerful tools for determining three-dimensional structures of biological or health-related compounds. Since the current procedures generally require years of effort, any technique which can expedite the process would be welcomed.